Improvement Of Nitrilase Regioselectivity And Its Application Research In Monocyanomonoacid Drug Intermediates

Compared with traditional chemical methods,enzyme-catalyzed reactions have attracted wide attention from experts and scholars in recent years due to their mild reaction conditions,excellent selectivity and environmentally friendly characteristics.Among them,several biocatalytic processes mediated by nitrilase have been successfully applied in many chemical fields instead of traditional chemical methods,including niacin,acrylic acid,and(R)-mandelic acid.Compared with chemical catalysts,nitrilase as a biocatalyst has advantages in reaction conditions and environmental fields,and more importantly,nitrilase exhibits excellent regional and stereoselectivity toward reaction substrates.Synthesis of monocyanomonoacid or diacid compounds based on the regio-and stereoselectivitycan be utilized as key high-value intermediates in the chemical and pharmaceutical industries.However,there is a lack of in-depth understanding of the regioselective mechanism of nitrilase,in addition to the wild nitrilases obtained naturally often fail to meet the requirements of regional or stereoselectivity,which severely limits the industrial application of nitrilases.In recent years,with the development of the computer technology,semi-rational engineering methods of enzyme molecules based on bioinformatics,structure and computational biology have gradually become the mainstream.In the present thesis,aiming at the above-mentioned problems faced by nitrilase,nitrilase from different sources and specific with dinitrile compounds are utlized as the research object,using the rational engineering methods including multi-sequence alignment,protein homology modeling,molecular docking,binding free energy calculation,etc.,starting from alanine scanning and polar scanning enzyme molecular modification strategies,combine with experimental techniques such as site-directed saturation and combined mutations to reveal the regioselective swithing mechanism toward the same substrate,the enhancing mechanism of substrate tolerance of nitrilase,the mechanism of product stereoselectivity enhancement and inversion.On this basis,identifying the key amino acid sites that regulate these characteristics,which provides a theoretical reference for the subsequent modification of other nitrilases to synthesize high-value chemical and pharmaceutical intermediates.The main research contents of the present thesis are as follows:In terms of the regulation and mechanism research of nitrilase regioselectivity switch,the difference analysis of the enzyme amino acid sequence and enzyme-substrate binding modes of two nitrilases from the same strain Bradyrhizobium japonicum USDA 110 were carried out based on protein homology modeling,multiple sequence alignment and molecular docking technology,and successfully identified the key sites Ala163 in b116402NIT and Trp172 in blr3397NIT of regulating regioselectivity.Using site-directed saturation mutation,by changing the distance(Dc-s)from the cyano carbon in the enzyme-substrate complex to the catalytic triad cysteine sulfhydryl sulfide(Dc-s),the size of the active center pocket is changed.Utilizing succinonitrile as the substrate,the catalytic products of nitrilase could be easily switched between 3-cyanopropionic acid and succinic acid as,laying a theoretical foundation for synthesizing regionally complementary monocyanomonoacid or diacid.Due to the low substrate tolerance and low conversion rate in terms of the process of nitrilase-mediated regioselective catalysis of terephthalonitrile to 4-cyano-benzoic acid,the classic alanine scanning mutation strategy and combined mutation were carried out molecularly modification of the nitrilase from Pseudomonas protegens strain pf5,and successfully obtained a mutant D200A/N256A with a substrate conversion rate increased by 1.2 folds,achieving 100%conversion rate with the substrate concentration from 50 mM to 300 mM.Theoretical studies revealed that the changes of key amino acids near the active center pocket of wild-type nitrilase lead to changes in hydrogen bonds,hydrophobic interactions,and ?-? non-bonding interactions between the substrate and nearby key amino acids compared with its mutants.The critical distance Dc-s changes from large to small,resulting in substarte tolerance improvement of the nitrilase.This study successfully identified the key sites to improve the tolerance of the nitrilase substrate,which can lay a theoretical foundation for the subsequent modification of the nitrilase to produce monocyanomono-4-cyano-benzoic acid.In terms of the regional and stereoselective resolution of racemic isobutyl succinonitrile to an important precursor(S)-3-cyano-5-methyl-hexanoic acid for the synthesis of pregabalin utilizing the nitrilase b116402NIT from Bradyrhizobium japonicum USDA 110,facing with the problem of a low product stereoselectivity(e.e=57.4%),the calculation method based on computer-aided MM/PBS A binding free energy was used to successfully identify the key amino acid sites Trp57 and Va1134 that can significantly enhance the stereoselectivity of the target product.Utilizing site-directed saturation and combination mutations,two mutants W57F/V134M and W57Y/V134M were obtained with high enantioselectivity(E>300).Both also showed extremely high e.e values(>99.9%)and conversion rates of 43.8%and 40.9%.Molecular docking and molecular dynamics simulation analysis showed that the potential mechanism of the significant enhancement of stereoselectivity is related to the change of the key distance parameter Dc-s and the formation of hydrogen bonds in the active center of nitrilase.The content of this chapter has successfully used the MM/PBS A method to identify the key amino acid sites that regulate the stereoselectivity of enzymes,and expands our deep mechanism of the nitrilase kinetic resolution reaction of racemic isobutyl succinonitrile.This understanding of nitrilase can not only provide theoretical guidance for the molecular modification of nitrilase,but also lay a solid foundation for the industrial production of optically pure pregabalin.In order to further obtain a highly stereoselective nitrilase that catalyzes the production of(S)-3-cyano-5-methyl-hexanoic acid,nitrilase NIT101 from Alcaligenes aquatilis with high product stereoselectivvity of(R)-3-cyano-5-methyl hexanoic acid(e.e=93%)was used as the research object.The "polar scanning" strategy was used to molecularly modification,combined with combined mutations,and successfully identified the key amino acid position Trpl87 for regulating stereoselective inversion of the product.A mutant W187N/S189N whose product configuration inverted from R-(e.e=93%)to S-(e.e=98.1%)was obtained.Molecular docking and molecular dynamics simulation analysis discovered that the critical distance between ?-cyanocarbon and the substrate sulfhydryl S-Dc-s changed significantly,and the hydrogen bonds of different configurations of substrates at the active center also changed,resulting in the change of product stereoselectivity.Based on the critical distance Dc-s,a streed molecular dynamics simulation was utilized to investisgate the ?-cyanocarbon in(R)-ISBN and(S)-ISBN in wild-type nitrilase and its mutants when substrates of different configurations enter the active pocket.The different orientations of the substrates lead to the preference for different configurations of the products.The content of this chapter not only provides important theoretical guidance for the subsequent modification of nitrilase to produce pregabalin intermediate(S)-3-cyano-5-methyl-hexanoic acid,taking an important step in its further practical application,but also provides a realistic example for realizing other enzymes to achieve product stereoselectivity inversion through the "polar scanning" strategy.Aiming at the unclear mechanism of regioselectivity regulation of nitrilase and the obvious problems in the production of high-value monocyanmonoacid intermediates,this thesis adopts the structural biology and computational biology methods,successfully revealed the regioselectivity switching mechanism of nitrilase to the same substrate,enhanced the substrate tolerance enhancement as well as enhancing and inverting products stereoselectivity.The key amino acid sites that regulate these characteristics are identified,and providing theoretical guidelines for the subsequent modifications of other nitrilase to synthesize high-value chemical and pharmaceutical monocyanmonoacid intermediates.